A number of immediate-early (IE) genes which constitute the primary genomic response to growth stimuli have been identified, including a novel gene, HLH462. HLH462 encodes a protein containing a helix-loop-helix (HLH) domain similar to that found in a class of transcriptional regulatory molecules important in cellular differentiation and embryonic development. The hypothesis that the protein product of the HLH462 gene plays an important role in the control of growth and development will be tested in four specific aims. First, the temporal and spatial pattern of HLH462 gene and protein expression in mouse embryos will be determined using the methods of in situ hybridization and immunohistochemistry with probes specific for HLH462. Second, specific antibody reagents will be generated and purified, and the biochemical and biological properties of the HLH462 protein will be investigated. Since the HLH462 gene structure predicts that the protein product contains an HLH domain which is known to be important for protein-protein interactions, the third aim of this proposal is to identify other known or unknown HLH proteins which are able to interact with the HLH462 protein. In vitro translated proteins derived from other cloned HLH genes will be tested for their ability to interact with purified recombinant HLH462 protein and for their ability to be co- immunoprecipitated with HLH462 antibodies. Proteins which interact with HLH462 in these assays will be further investigated. Genes encoding uncharacterized proteins which can interact with HLH462 can be cloned by using labeled recombinant HLH462 protein as a molecular probe. The fourth aim of the proposed research is to test the hypothesis that HLH462 protein is necessary for growth regulation in cultured cells. The normal pattern of expression of HLH462 in these cells will be disturbed in order to assay the effects on growth and the response to growth stimulation. To inhibit the induction of HLH462, antisense RNA expression constructs and antisense oligonucleotides will be employed. In addition, anti-HLH462 antibodies will be injected into cells in order to neutralize the activity of the protein. The ability of overexpressed HLH462 protein to confer a growth advantage or disadvantage on fibroblast cells will also be assayed. It is expected that these experiments will determine whether the induction of HLH462 (a growth-associated gene) is necessary for cell growth. The determination of the sites of HLH462 expression, characterization of the protein's properties, identification of protein partners and determination of the effects of HLH462 on cell growth regulation will be important for understanding the role of HLH462 in the control of normal and abnormal growth and development and disease states which involve regulation of these processes.